Biology subjects

Benfey, P. N.

Publications and source records attributed to Benfey, P. N..

At least 19 recordsLinked to original sources

bioRxiv2025

GeneSys: Generative Modeling of Developmental System

Temporal single-cell transcriptomics enables the reconstruction of dynamic gene expression changes during development. Yet, its analytical power is often limited by data sparsity, technical noise, and imbalanced representation of cell types across time points. To overcome these challenges, we present GeneSys (Generative Modeling of Developmental System), a generative deep learning model that simulates single-cell transcriptomic landscapes under developmental constraints, which is informed by prior biological knowledge or user-defined hypotheses. GeneSys integrates a temporal variational autoencoder with a cell-type classifier, requiring a lineage blueprint as input, which enables it to model the temporal transitions of transcriptional states with cell-type specificity. Leveraging data from Arabidopsis thaliana roots and mouse embryos, we show that GeneSys learns robust developmental trajectories, generates imputed and representative transcriptomes, and enhances gene prioritization accuracy compared to unregularized scRNA-seq data. By applying gene masking and augmentation, GeneSys reveals interpretable gene expression programs (GEPs) and serves as an in silico platform to test the impact of specific genes or gene sets on user-defined developmental outcomes. Additionally, GeneSys computes linear interaction matrices (LIMAs) to infer dynamic gene networks and prioritize transcription factors with spatiotemporal resolution. These features enable GeneSys to nominate key genes governing state transitions in a developmental system, supporting both mechanistic insight and hypothesis generation. Together, GeneSys provides a flexible and extensible framework to denoise single-cell data and simulate transcriptomic developmental landscape guided by known or hypothesized developmental constraints, empowering the discovery of regulatory mechanisms from high-dimensional single-cell datasets.

systems biology↗

bioRxiv2022

Patterning and growth are coordinated early in the cell cycle

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative (asymmetric) divisions that generate new tissue patterns with proliferative (symmetric) divisions that promote growth is poorly understood. Here, we employed quantitative 4D light sheet and confocal microscopy to probe in vivo the dynamics of two transcription factors, SHORTROOT (SHR) and SCARECROW (SCR), which are required for asymmetric division in the stem cell niche of Arabidopsis roots [1,2]. Long-term (up to 48 hours), frequent (every 15 minutes) imaging of the two regulators in tandem in single cells, in conjunction with a SHR induction system, enabled us to challenge an existing bistable model[3] of the SHR/SCR gene regulatory network. By directly controlling SHR and SCR expression dynamics, we were able to identify key features that are essential for rescue of asymmetric division in shr mutants. We show that instead of high stable levels of nuclear SHR and SCR, only low transient levels of expression are required. Nuclear SHR kinetics do not follow predictions of the bistable model, and the regulatory relationship between SHR and SCR can be modeled by monostable alternatives. Furthermore, expression of these two regulators early in the cell cycle determines the orientation of the division plane, resulting in either formative or proliferative cell division. Our findings provide evidence for an uncharacterized mechanism by which developmental regulators directly coordinate patterning and growth.

developmental biology↗

bioRxiv2022

Brassinosteroid gene regulatory networks at cellular resolution

Brassinosteroids (BRs) are plant steroid hormones that regulate diverse processes such as cell division and cell elongation. BRs control thousands of genes through gene regulatory networks that vary in space and time. By using time-series single-cell RNA-sequencing to identify BR-responsive gene expression specific to different cell types and developmental stages of the Arabidopsis root, we uncovered the elongating cortex as a site where BRs trigger a shift from proliferation to elongation associated with increased expression of cell wall-related genes. Our analysis revealed HAT7 and GTL1 as BR-responsive transcription factors that regulate cortex cell elongation. These results establish the cortex as an important site for BR-mediated growth and unveil a BR signaling network regulating the transition from proliferation to elongation, illuminating new aspects of spatiotemporal hormone response.

plant biology↗

bioRxiv2022

Single-cell transcriptomics of the Arabidopsis floral abscission zone

Abscission is the programmed separation of plant organs. It is widespread in the plant kingdom with important functions in development and environmental response. In Arabidopsis, abscission of floral organs (sepals, petals, and stamens) is controlled by two receptor-like protein kinases HAESA (HAE) and HAESA LIKE-2 (HSL2), which orchestrate the programmed dissolution of the abscission zone connecting floral organs to the developing fruit. In this work, we use single-cell RNA-Sequencing to characterize the core HAE/HSL2 abscission gene expression program. We identify the MAP KINASE PHOSPHATASE-1/MKP1 gene as a negative regulator of this pathway. MKP1 acts prior to activation of HAE/HSL2 signaling to establish a signaling threshold required for the initiation of abscission. By analogy to electrical circuit control, we liken MKP1 to a molecular grounding mechanism that dissipates errant pathway activation absent HAE/HSL2 signaling. Furthermore, we use single-cell data to identify genes expressed in two sub-populations of abscission zone cells: those proximal and those distal to the plane of separation. We identify INFLORESCENCE DEFICIENT IN ABSCISSION/IDA, encoding the activating ligand of HAE/HSL2, as one of the mRNAs most highly enriched in distal abscission zone cells at the base of the abscising organs. We show how this expression pattern forms a biophysical circuit breaker whereby, when the organ is shed, the source of the IDA peptide is removed, leading to cessation of HAE/HSL2 signaling. Overall, this work provides insight into the multiple control mechanisms acting on the abscission-signaling pathway.

plant biology↗

bioRxiv2022

Cell-type specific transcriptomics reveals roles for root hairs and endodermal barriers in interaction with beneficial rhizobacterium

Growth-promoting bacteria can boost crop productivity in a sustainable way. Pseudomonas simiae WCS417 is a well-studied bacterium that promotes growth of many plant species. Upon colonization, WCS417 affects root system architecture resulting in an expanded root system. Both immunity and root system architecture, are controlled by root-cell-type specific biological mechanisms, but it is unknown how WCS417 affects these mechanisms. Therefore, here, we transcriptionally profiled five Arabidopsis thaliana root cell types following WCS417 colonization. The cortex and endodermis displayed the most differentially expressed genes, even though they were not in direct contact with this epiphytic bacterium. Many of these genes are associated with reduced cell wall biogenesis, possibly facilitating the root architectural changes observed in WCS417-colonized roots. Comparison of the transcriptome profiles in the two epidermal cell types that were in direct contact with WCS417 - trichoblasts that form root hairs and atrichoblasts that dont - imply functional specialization. Whereas basal expression levels of nutrient uptake-related genes and defense-related genes are highest in trichoblasts and atrichoblasts, respectively, upon exposure to WCS417 these roles revert. This suggests that root hairs participate in the activation of root immunity, further supported by attenuation of immunity in a root hairless mutant. Furthermore, we observed elevated expression of suberin biosynthesis genes and increased deposition of suberin in the endodermis in WCS417-colonized roots. Using an endodermal barrier mutant we show the importance of endodermal barrier integrity for optimal plant-beneficial bacterium association. Altogether, we highlight the strength of cell-type-specific transcriptional profiling to uncover "masked" biological mechanisms underlying successful plant-microbe associations.

plant biology↗

bioRxiv2022

The rhizodynamics robot: Automated imaging system for studying long-term dynamic root growth

The study of plant root growth in real time has been difficult to achieve in an automated, high-throughput, and systematic fashion. Dynamic imaging of plant roots is important in order to discover novel root growth behaviors and to deepen our understanding of how roots interact with their environments. We designed and implemented the Generating Rhizodynamic Observations Over Time (GROOT) robot, an automated, high-throughput imaging system that enables time-lapse imaging of 90 containers of plants and their roots growing in a clear gel medium over the duration of weeks to months. The system uses low-cost, widely available materials. As a proof of concept, we employed GROOT to collect images of orchid root growth of multiple species over six months. Beyond imaging plant roots, our system is highly customizable and can be used to collect time-lapse image data of different container sizes and configurations regardless of what is being imaged, making it applicable to many fields that require longitudinal time-lapse recording.

plant biology↗

bioRxiv2021

Plant immune system activation is necessary for efficient interaction with auxin secreting beneficial bacteria

Plants continuously monitor the presence of microorganisms through their immune system to establish an adaptive response. Unlike immune recognition of pathogenic bacteria, how beneficial bacteria interact with the plant immune system is not well understood. Analysis of colonization of Arabidopsis thaliana by auxin producing beneficial bacteria revealed that activation of the plant immune system is necessary for efficient bacterial colonization and auxin secretion. A feedback loop is established in which bacterial colonization triggers an immune reaction and production of reactive oxygen species, which, in turn, stimulate auxin production by the bacteria. Auxin promotes bacterial survival and efficient root colonization, allowing the bacteria to inhibit fungal infection and promote plant health.

plant biology↗

bioRxiv2020

Capturing in-field root system dynamics with the RootTracker

Optimizing root system architecture offers a promising approach to developing stress tolerant cultivars in the face of climate change, as root systems are critical for water and nutrient uptake as well as mechanical stability. However, breeding for optimal root system architecture has been hindered by the difficulty in measuring root growth in the field. Here, we describe a technology, the RootTracker (RT), which employs capacitance touch sensors to monitor in-field root growth over time. Configured in a cylindrical shutter-like fashion around a planted seed, 264 electrodes are individually charged multiple times over the course of an experiment. Signature changes in the measured capacitance and resistance readings indicate when a root has touched or grown close to an electrode. Using the RootTracker, we have measured root system dynamics of commercial maize hybrids growing in both typical Midwest field conditions and under different irrigation regimes. We observed rapid responses of root growth to water deficits and found evidence for a "priming response" in which an early water deficit causes more and deeper roots to grow at later time periods. There was genotypic variation among hybrid maize lines in their root growth in response to drought, indicating a potential to breed for root systems adapted for different environments.

plant biology↗

bioRxiv2020

A plant lipocalin is required for retinal-mediated de novo root organogenesis

Branching of root systems enables the exploration and colonization of the soil environment. In Arabidopsis roots, de novo organogenesis of lateral roots is patterned by an oscillatory mechanism called the root clock, which is dependent on metabolites derived from the {beta}-carotene pathway1, 2. Retinoids are {beta}-carotene-derived regulators of organogenesis in the animal kingdom. To determine if retinoids function in plant development, we conducted time-lapse imaging of a chemical reporter for retinoid binding proteins. We found that it oscillates with a comparable frequency to the root clock and accurately predicts sites of lateral root organogenesis. Exogenous application of retinal to wild-type plants is sufficient to induce root clock oscillations and lateral root organogenesis. A homology search yielded a potential Arabidopsis homolog, TEMPERATURE INDUCED LIPOCALIN (TIL) to vertebrate retinoid binding proteins. Genetic analysis indicates that TIL is necessary for normal lateral root development and a til mutant has decreased retinal sensitivity. TIL expression in a heterologous system conferred retinal binding activity, suggesting that it may directly interact with this molecule. Together, these results demonstrate an essential role for retinal and for plant retinal binding proteins in lateral root organogenesis.

plant biology↗

bioRxiv2020

A single cell Arabidopsis root atlas reveals developmental trajectories in wild type and cell identity mutants

SummaryCell fate acquisition is a fundamental developmental process in all multicellular organisms. Yet, much is unknown regarding how a cell traverses the pathway from stem cell to terminal differentiation. Advances in single cell genomics1 hold promise for unraveling developmental mechanisms2–3 in tissues4, organs5–6, and organisms7–8. However, lineage tracing can be challenging for some tissues9 and integration of high-quality datasets is often necessary to detect rare cell populations and developmental states10,11. Here, we harmonized single cell mRNA sequencing data from over 110,000 cells to construct a comprehensive atlas for a stereotypically developing organ with indeterminate growth, the Arabidopsis root. To test the utility of the atlas to interpret new datasets, we profiled mutants for two key transcriptional regulators at single cell resolution, shortroot and scarecrow. Although both transcription factors are required for early specification of cell identity12, our results suggest the existence of an alternative pathway acting in mature cells to specify endodermal identity, for which SHORTROOT is required. Uncovering the architecture of this pathway will provide insight into specification and stabilization of the endodermis, a tissue analogous to the mammalian epithelium. Thus, the atlas is a pivotal advance for unraveling the transcriptional programs that specify and maintain cell identity to regulate organ development in space and time.Competing Interest StatementPNB is the co-founder and Chair of the Scientific Advisory Board of Hi Fidelity Genetics, Inc, a company that works on crop root growth.View Full Text

plant biology↗

bioRxiv2020

VST Family Proteins are Regulators of Root System Architecture in Rice and Arabidopsis

Root System Architecture (RSA) is a key factor in the efficiency of nutrient capture and water uptake in plants. Understanding the genetic control of RSA will be useful in minimizing fertilizer and water usage in agricultural cropping systems. Using a hydroponic screen and a gel-based imaging system we identified a rice gene, OsVST1, which plays a key role in controlling RSA. This gene encodes a homolog of the Arabidopsis VAP-RELATED SUPPRESSORS OF TMM (VSTs), a class of proteins that promote signaling in stomata by mediating plasma membrane-endoplasmic reticulum contacts. OsVST1 mutants have shorter primary roots, decreased root meristem size, and a more compact root system architecture. We show that the Arabidopsis VST triple mutants have similar phenotypes, with reduced primary root growth and smaller root meristems. Expression of OsVST1 largely complements the short root length and reduced plant height in the Arabidopsis triple mutant, supporting conservation of function between rice and Arabidopsis VST proteins. In a field trial, mutations in OsVST1 do not adversely affect grain yield, suggesting that modulation of this gene could be used as a way to optimize RSA without an inherent yield penalty.

plant biology↗

bioRxiv2020

Mechanism and function of root circumnutation

AbstractEarly root growth is critical for plant establishment and survival. We have identified a molecular pathway required for oscillatory root tip movement known as circumnutation. Here we report a multiscale investigation of the regulation and function of this phenomenon. We identify key cell signaling events comprising interaction of the ethylene, cytokinin, and auxin hormone signaling pathways. We identify the gene Oryza sativa Histidine Kinase-1/OsHK1, as well as the auxin influx carrier gene OsAUX1, as essential regulators of this process in rice. Robophysical modelling demonstrated the benefits of tip movement for navigating past obstacles, prompting us to challenge mutant and wild-type plants with different substrates. Consistent with model behavior, root circumnutation facilitated exploration of a solid surface and promoted seedling establishment in rocky soil. Thus, the integration of robotics, physics and biology elucidated the functional importance of root circumnutation and uncovered the molecular mechanisms underlying its regulation. One sentence summaryCircumnutation facilitates root exploration.

plant biology↗

bioRxiv2020

Cell wall remodeling and vesicle trafficking mediate the root clock in Arabidopsis

In Arabidopsis, lateral roots initiate along the primary root in a process preceded by periodic gene expression, a phenomenon known as the root clock. Many genes involved in lateral root initiation have been identified. However, very little is known about the structural changes underlying the initiation process nor about how root clock function is regulated. In genetic screens, we identified the vesicle trafficking regulators, GNOM and its suppressor, AGD3, as critical to root clock function. We show that GNOM is required for the proper distribution of pectin, a mediator of intercellular adhesion, and that pectin esterification state is essential for a functional root clock. We found that in sites of lateral root primordia emergence, both esterified and de-esterified pectin are differentially distributed. Using a reverse genetic approach, we identified significant enrichment of GO terms associated with pectin modifying enzymes in the oscillation zone were the root clock is established. In agreement with a recent study on the function of pectin in pavement cell morphogenesis, our results indicate that the balance between esterified and de-esterified pectin is essential for proper root clock function and the subsequent initiation of lateral root primordia.

plant biology↗

bioRxiv2018

Anchorene is an endogenous diapocarotenoid required for anchor root formation in Arabidopsis

Arabidopsis root development is predicted to be regulated by yet unidentified carotenoid-derived metabolite(s). In this work, we screened known and putative carotenoid cleavage products and identified anchorene, a predicted carotenoid-derived dialdehyde (diapocarotenoid) that triggers anchor root development. Anchor roots are the least characterized type of root in Arabidopsis. They form at the root-shoot junction, particularly upon damage to the root apical meristem. Using Arabidopsis reporter lines, mutants and chemical inhibitors, we show that anchor roots originate from pericycle cells and that the development of this root type is auxin-dependent and requires carotenoid biosynthesis. Transcriptome analysis and treatment of auxin-reporter lines indicate that anchorene triggers anchor root development by modulating auxin homeostasis. Exogenous application of anchorene restored anchor root development in carotenoid-deficient plants, indicating that this compound is the carotenoid-derived signal required for anchor root development. Chemical modifications of anchorene led to a loss of anchor root promoting activity, suggesting that this compound is highly specific. Furthermore, we demonstrate by LC-MS analysis that anchorene is a natural, endogenous Arabidopsis metabolite. Taken together, our work reveals a new member of the family of carotenoid-derived regulatory metabolites and hormones.\n\nSignificanceUnknown carotenoid-derived compounds are predicted to regulate different aspects of plant development. Here, we characterize the development of anchor roots, the least characterized root type in Arabidopsis, and show that this process depends on auxin and requires a carotenoid-derived metabolite. We identified a presumed carotenoid-derivative, anchorene, as the likely, specific signal involved in anchor root formation. We further show that anchorene is a natural metabolite that occurs in Arabidopsis. Based on the analysis of auxin reporter lines and transcriptome data, we provide evidence that anchorene triggers the growth of anchor roots by modulating auxin homeostasis. Taken together, our work identifies a novel carotenoid-derived growth regulator with a specific developmental function.

plant biology↗

bioRxiv2018

A histidine kinase gene is required for large radius root tip circumnutation and surface exploration in rice

The intricate growth patterns that accompany plant organ elongation have long intrigued biologists 1. Circumnutation refers to the circular or elliptical growth of the tip of a plant organ around a central axis. While the utility of circumnutation for climbing plants is clear, its function in roots is less obvious. Additionally, the genetic requirements for root circumnutation are not known. Here we show that mutations in a gene encoding a histidine kinase abolish large radius root tip circumnutation in rice. Using a gel-based imaging system and a whole genome sequenced mutant population, we identified three different mutant alleles of the gene OsHK1 that exhibit increased seedling root depth. Time-lapse imaging indicated that this phenotype is likely due to a lack of large radius root tip circumnutation in OsHK1 mutants. Treatment of mutant roots with the plant hormone zeatin rescues circumnutation, indicating that OsHK1 functions in a cytokinin-related signaling pathway. We found that OsHK1 mutants are impaired in their ability to explore flat surfaces, suggesting that circumnutation facilitates root exploration at the interface of compacted soil horizons.

plant biology↗

bioRxiv2018

β-cyclocitral is a natural root growth regulator

Natural compounds capable of increasing root depth and branching are desirable tools for enhancing stress tolerance in crops. We devised a sensitized screen to identify natural metabolites capable of regulating root traits in Arabidopsis. {beta}-cyclocitral, an endogenous root compound, was found to promote cell divisions in root meristems and stimulate lateral root branching. {beta}-cyclocitral rescued meristematic cell divisions in ccd1ccd4 biosynthesis mutants and {beta}-cyclocitral-driven root growth was found to be independent of auxin, brassinosteroid, and ROS signaling pathways. {beta}-cyclocitral had a conserved effect on root growth in tomato and rice and generated significantly more compact crown root systems in rice. Moreover, {beta}-cyclocitral treatment enhanced plant vigor in rice plants exposed to salt-contaminated soil. These results indicate that {beta}-cyclocitral is a broadly effective root growth promoter in both monocots and eudicots and could be a valuable tool to enhance crop vigor under environmental stress.\n\nOne Sentence Summary{beta}-cyclocitral is a metabolite of {beta}-carotene that was identified using a sensitized chemical screen and acts broadly across plants to enhance root growth and branching.

developmental biology↗

bioRxiv2018

Root meristem growth factor 1 controls root meristem size through reactive oxygen species signaling

Stem cell niche and root meristem size are maintained by intercellular interactions and signaling networks of a plant peptide hormone, Root Meristem Growth Factor 1 (RGF1). How RGF1 regulates root meristem development is an essential question to understand stem cell function. Although five receptors of RGF1 have recently been identified, the downstream signaling mechanism remains unknown. Here, we report a series of signaling events following RGF1 action. The RGF1-receptor pathway controls distribution of reactive oxygen species (ROS) along the developmental zones of the Arabidopsis root. We identify a novel transcription factor, RGF1 INDUCIBLE TRANSCRIPTION FACTOR 1 (RITF1), which plays a central role in mediating RGF1 signaling. Manipulating RITF1 expression leads to redistribution of ROS along the root developmental zones. Changes in ROS distribution, in turn, enhance the stability of the PLETHORA2 (PLT2) protein, a master regulator of root stem cells. Taken together, our study clearly depicts a signaling cascade initiated by RGF1 and links the RGF1 peptide to ROS regulatory mechanisms.

plant biology↗

bioRxiv2017

Minimum requirements for reprogramming and maintaining cell fate in the Arabidopsis root

Changes in gene regulation during differentiation are governed by networks of transcription factors. To identify the minimal network for endodermal differentiation in the Arabidopsis root, we asked what transcription factors are sufficient to program a non-native cell-type into endodermis. Our results show the transcription factors SHORTROOT and MYB36 have limited ability to reprogram a non-native cell-type (the epidermis) and that this reprogramming is reversible in the absence of additional cues. The stele-derived signaling peptide CIF2 stabilizes SHORTROOT-induced reprogramming. The outcome is a partially impermeable barrier deposited in the sub-epidermal cell layer that has a transcriptional signature similar to endodermis. The trans-differentiation mechanism depends on the expression of genes downstream in the gene regulatory network but is independent of SHORTROOT movement. These results highlight a non cell-autonomous induction mechanism for endodermis that resembles differentiation in many animal systems.\n\nOne Sentence SummarySHORTROOT and CIF2 combined can induce and stabilize an endodermis in sub-epidermal cells and do so in a non cell-autonomous manner.

developmental biology↗